Interspecific nicotiana hybrids and their progeny

ABSTRACT

The present invention relates to novel interspecific  Nicotiana excelsior  x  N. benthamiana  hybrid seeds and plants and to a method of producing interspecific Nicotiana hybrids having enhanced properties for biomass and the production of recombinant proteins using a viral vector system.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of continuation-in-partapplication Ser. No. 09/353,787 filed Jul. 15, 1999 which is acontinuation-in-part of application Ser. No. 09/232,170 filed on Jan.15, 1999, which in turn is a continuation-in-part application of Ser.No. 09/008,186 filed on Jan. 16, 1998, each incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to novel interspecific Nicotianaexcelsior x N. benthamiana hybrid seeds and plants and to a method ofproducing interspecific Nicotiana hybrids having enhanced properties forbiomass and the production of recombinant proteins using a viral vectorsystem.

[0003] The difference between intra- and interspecific hybrids can bevisualized by the Mendelian behavior of intraspecific hybrids and theessentially non-Mendelian behavior of interspecific hybrids. Thisdifference results from the complete chromosome homology in hybridsbetween plants of the same species (intraspecific) and the partial ornonchromosome homology that characterizes hybrids between plants ofdifferent species (interspecific). The intermediate or gray area betweenthe extremes among interspecific hybrids is seen in the behavior ofthose involving closely related species. Such species will have the samechromosome number, and the extent of chromosome homologies may be veryhigh. Hybrids of this kind produce seed when self-pollinated and showevidence of Mendelian patterns of segregation for some traits.

[0004] Cytological studies of meiosis in some F₁ hybrids may showevidence of chromosome irregularities that reflect the chromosomaldifferences that mark the parents as different species. A hybrid betweendistantly related species may show reduced pairing between chromosomesof the different genomes. Meiosis in such hybrids may exhibit thetypical chromosome behavior characteristic of monogenomic haploids. Mostinterspecific F₁ hybrid combinations in the genus Nicotiana fall intothis latter category. Their chromosome doubled counterparts are calledamphidiploids, or simply allopolyploids, when a parent of the hybrid mayitself be a combination of different ancestral genomes. N. tabacumrepresents a typical example.

[0005] The relative difficulty of producing interspecific F₁ hybridsincreases in proportion to the decrease in taxonomic relations betweentheir parents. Although some F₁ hybrids are stable when converted tofertile polyploids, even hybrids between distantly related parents sharesome degree of chromosome homology. There are advantages to maintainingseed stocks of allopolyploids by self-pollination, particularly if thehybrid is difficult to obtain by conventional cross-pollination.

[0006] Interspecific hybridization and introgression in naturalpopulations of plants and animals is a known source of genetic variationand adaptation. The term introgression, or introgressive transfer, ofgenetic traits is used in a narrower sense with short-term breedingprojects. The goal of experimental interspecific hybridization isusually the introgressive transfer of a specific trait from one species(the nonrecurrent parent) into the genome of another (the recurrentparent). The trait must be expressed with reasonable phenotypic fidelityas a dominant or partial dominant in the F₁ hybrid and all subsequentbackcross generations, when nonrecurrent chromosomes are lost at random,otherwise the proposed interspecific transfer could not be made. Foreigngenes from one species can become unstable when translocated into thegermplasm of another.

[0007] Chromosome pairing between the foreign genomes in aninterspecific hybrid may be extensive or minimal although the F₁ hybridis usually sterile. Fertility may be restored by treating germinatingseed of the hybrid in 0.4 percent aqueous colchicine for about 4 hoursto induce chromosome doubling. Immersion of the seeds in 0.1 percentcolchicine for 24 hours may also be effective. Thereafter, the seed isrinsed in sterile distilled water and planted by sowing on the surfaceof pasteurized soil in glass or plastic preparation dishes. Theseedlings later are transplanted to pots of soil in the greenhouse andmature plants that show good pollen development are self pollinated orbackcrossed to the recurrent parent.

[0008] Three conventional approaches to interspecific hybridization forthe purpose of alien transfer and incorporation of germplasm into stablediploid lines are (a) diploid x diploid then doubling to produce theallopolyploid and backcrossing to produce the sesquidiploid, (b)autotetraploid x autotetraploid to produce the allopolyploid directly,and (c) autotetraploid x diploid to produce the sesquidiploid directlyas described in Technical Bulletin 1586, U.S. Department of Agriculture,1979.

[0009] Gene transfer mediated by Agrobacterium tumefaciens vectors hasbecome routine in tobacco (DeBlock et al., 1987; Grierson, et al., 1990;Hilder, et al., 1990; Lindbo and Dougherty, 1992). Commercial use ofgenetic transformation in agriculture depends on the incorporation offoreign genes into high-yielding germplasm. Plant breeders may want tocombine several foreign genes into a single elite germplasm source. Inorder to accomplish this goal, designer chromosome construction isdesirable.

[0010] Designer, or artificial, chromosomes have been produced in yeast.In plants, the scaffold for designer chromosome construction can befound in a breeding line that possesses the full complement ofchromosomes from its own species plus an addition chromosome from arelated species, Campbell, et al., Construction of a designer chromosomein tobacco, Theor Appl Genet (1994) 87:837-842. Additional chromosomesare often meiotically stable as homozygotes, and because recombinationbetween additional chromosomes and the rest of the plant genome is rare(Gerstel 1945) the integrity of a foreign-gene linkage package can bepreserved.

[0011] The placement of this linkage package on an additional chromosomewill also minimize disturbance to the rest of the plant genome. Thegenetic structure of high-yielding germ plasm can be disturbed eitherthrough insertional mutagenesis or the disturbance of beneficial linkageblocks. Insertional mutagenesis, caused by the integration of foreigngenes into plant coding sequences, can occur frequently. Konez, et al.(1989) estimated that at least 30% of all T-DNA insertions occur intranscribed regions of the Arabidopsis and Nicotiana genomes. Beneficiallinkage blocks are formed through intermating and recombination followedby selection.

[0012] The desired traits for a plant may not be present in thegermplasm of the species of interest. In such a case, traditionalbreeding within the species may not give acceptable results. Introducinga desired trait from one species into a related one by interspecifichybridization is followed by introgression into the recurrent parent.

[0013] Methods for expressing genes in plants has been described in U.S.Pat. Nos. 5,316,931; 5,589,367; 5,811,653 and 5,866,785, all of whichare incorporated herein. Certain species have either an acceptablebiomass or an acceptable expression of a sequence inserted into a plantviral vector, but no current species have both high levels of biomassand high levels of viral vector performance. There is a need for amethod to develop in one species both the characteristics of increasedbiomass and improved viral vector performance.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to a method of crossing twodifferent species of Nicotiana to produce an interspecific hybrid whichresults in an increased biomass and improved host susceptibility to aviral vector.

[0015] In accordance with this invention, interspecific Nicotianahybrids have been produced that are characterized with having a biomassof greater than that usually obtained from tobacco with CP-fusionconstructs and a virion yield of greater than 1.0 mg. virion/gram freshweight.

[0016] In one embodiment of the invention, there is provided a novelNicotiana interspecific hybrid. This invention thus relates to the seedsof Nicotiana interspecific hybrid, to the plants of Nicotianainterspecific hybrid and to methods for producing a Nicotianainterspecific hybrid plant produced by crossing the Nicotianainterspecific hybrid with itself or another Nicotiana interspecifichybrid line.

[0017] Thus, any such methods using the Nicotiana interspecific hybridare part of this invention: selfing, backcrosses, hybrid production,crosses to populations, and the like. All plants produced using aNicotiana interspecific hybrid as a parent are within the scope of thisinvention. Advantageously, the Nicotiana interspecific hybrid could beused in crosses with other, different, Nicotiana interspecific hybridsto produce first generation (F₁) Nicotiana interspecific hybrid seedsand plants with superior characteristics.

[0018] In another aspect, the present invention provides for single geneconverted plants of Nicotiana interspecific hybrid. The singletransferred gene may preferably be a dominant or recessive allele.Preferably, the single transferred gene will confer such traits asherbicide resistance, insect resistance, resistance for bacterial,fungal, or viral disease, male fertility, male sterility, enhancednutritional quality, and industrial usage. The single gene may be anaturally occurring Nicotiana gene or a transgene introduced throughgenetic engineering techniques.

[0019] In another aspect, the present invention provides regenerablecells for use in tissue culture of a Nicotiana interspecific hybrid. Thetissue culture will preferably be capable of regenerating plants havingthe physiological and morphological characteristics of the foregoingNicotiana interspecific hybrid, and of regenerating plants havingsubstantially the same genotype as the foregoing Nicotiana interspecifichybrid. Preferably, the regenerable cells in such tissue cultures willbe embryos, protoplasts, meristematic cells, callus, pollen, leaves,anthers, roots, root tips, flowers, seeds, pods or stems. Still further,the present invention provides Nicotiana plants regenerated from thetissue cultures of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0020] To provide an understanding of several of the terms used in thespecification and claims, the following definitions are provided:

[0021] Geneware™ viral vector—As used herein, Geneware™ viral vectorrefers to a system for expressing genes in plants, such as described inU. S. Pat. Nos. 5,316,931; 5,589,367; 5,811,653 and 5,866,785, allincorporated herein by reference.

[0022] Biomass—As used herein, the term “biomass” means the total freshweight in grams of plant tissue which is harvested above the ground. Theterms “fresh weight” and “biomass” are used interchangeably herein.

[0023] Virion yield—As used herein the term “virion yield” means thetotal yield of the virus measured in mg of virion per gram fresh weight.

[0024] Host—As used herein, a “host” means a cell, tissue or organismcapable of supporting replication of a vector or plant viral nucleicacid and which is capable of being infected by a virus containing theviral vector or plant viral nucleic acid.

[0025] Infection—As used herein, an “infection” means the ability of avirus to transfer its nucleic acid to a host or introduce viral nucleicacid into a host, wherein the viral nucleic acid is replicated, viralproteins are synthesized, and new viral particles assembled. In thiscontext, the terms “transmissible” and “infective” are usedinterchangeably herein.

[0026] Phenotypic Trait—As used herein, “phenotypic trait” is anobservable property resulting from the expression of a gene or genes.

[0027] Plant Tissue—As used herein, “plant tissue” is any tissue of aplant in planting or in culture. This term is intended to include awhole plant, plant cell, plant organ, protoplast, cell culture, or anygroup of plant cells organized into a structural and functional unit.

[0028] Recombinant Plant Viral Nucleic Acid—As used herein, “recombinantplant viral nucleic acid” means it has been modified to containnonnative nucleic acid sequences.

[0029] Recombinant Plant Virus—As used herein, “recombinant plant virus”is a plant virus containing the recombinant plant viral nucleic acid.

[0030] Vector—As used herein, “vector” is a self-replicating nucleicacid molecule which transfers a nucleic acid segment between cells.

[0031] Virus—As used herein, “virus” is an infectious agent composed ofa nucleic acid encapsidated in protein. A virus may be a mono-, di-,tri- or multi-partite virus.

[0032] Backcrossing—As used herein, “backcrossing” is a process in whicha breeder repeatedly crosses hybrid progeny back to one of the parents,for example, a first generation hybrid F₁ with one of the parentalgenotypes of the F₁ hybrid.

[0033] Essentially all the physiological and morphologicalcharacteristics—As used herein, a plant having essentially all thephysiological and morphological characteristics means a plant having thephysiological and morphological characteristics, except for thecharacteristics derived from the converted gene.

[0034] Single Gene Converted (Conversion)—As used herein, “single geneconverted (conversion)” plant refers to plants which are developedwherein essentially all of the desired morphological and physiologicalcharacteristics of a variety are recovered in addition to the singlegene transferred into the variety via the backcrossing technique or viagenetic engineering.

[0035] Trichomes—As used herein, “trichomes” means the glandular hairson the surface of the plant stem, leaf, calyx, corolla or other planttissue surfaces.

[0036] Leaf lamina—As used herein, “leaf lamina” means the leaf surfacedevoid of vascular tissue of primary, secondary or ternary nature.

[0037] GFP-GJ—As used herein, “GFP-GJ” means a crude plant extractcontaining green fluorescent protein.

[0038] GFP-pH5, Δ—As used herein, “GFP-pH5, Δ” means a crude plantextract that was treated with heat and a pH of 5 that contains greenfluorescent protein that is enriched for its presence.

[0039] CP-GJ—As used herein, “CP-GJ” means a crude plant extractcontaining viral coat protein.

[0040] CP-pH5, Δ—As used herein, “CP-pH5, Δ” means a crude plant extractthat was treated with heat and a pH of 5 that contains coat protein thatis enriched for its presence.

[0041] Host plant species vary in their ability to support expression ofa sequence inserted into a plant viral vector. For example, some species(such as Nicotiana benthamiana) support expression from adual-subgenomic promoter tobamoviral vector to a high specific activity,but have relatively low biomass. Other species (such as N. tabacum) havehigh biomass and/or other desirable properties for growth in the field,but have a relatively low specific activity of the expressed protein.Using the method of the present invention, after chromosome doubling torestore fertility, the primary hybrid may have suitable properties, orit may be desirable to backcross toward either parent selecting orscreening at each generation for the desired properties of thenon-recurrent parent (e.g., introgressing the superior biomass of N.tabacum into N. benthamiana, or introgressing the superior viral vectorperformance of N. benthamiana into N. tabacum). A viral vectorexpressing the green fluorescent protein (GFP) is one example of auseful tool for screening the level of systemic expression in candidatehybrid plants. Geneware™ viral vector refers to a system for expressinggenes in plants such as described in U.S. Pat. Nos. 5,316,931;5,589,367; 5,811,653 and 5,866,785, all incorporated herein byreference.

EXAMPLES

[0042] The following examples are provided to further illustrate thepresent invention and are not intended to limit the invention beyond thelimitations set forth in the appended claims.

Example 1 Procedures for Making Interspecific Crosses of Nicotiana

[0043] When making interspecific crosses it is important to have: 1)healthy plants, 2) careful emasculation, 3) freedom from contaminationby foreign pollen, 4) a reasonable level of cross fertility among theparent plants, and 5) recording of the crosses. A pair of finecurved-point forceps is used to slit the corolla and emasculate theblossom. In the case of N. tabacum, and many other species, the anthersare removed just before the corolla unfolds. Several exceptions apply tothis general rule. In the case of N. repanda, the anthers are adnate tothe stigma, the anther walls usually break down, and the contents of theanther adhere to the stigma before the flower matures. Anthers in thelater-formed flowers of N. nudicaulis may dehisce before the corollaunfolds and self-pollination will occur. The flowers of these speciesare emasculated several days before cross-pollinations are made.

[0044] Ordinarily, pollinations are made immediately after emasculation,up to 18 to 24 hours after emasculation before applying pollen. Theglistening and sticky surface of the stigma is touched with a newlydehisced anther, or pollen may be applied with the aid of a smallartist's brush (size 6/128 to 10/128). Brush pollination aids seed setin some self-fertile species, and sib-pollinations are required toproduce seed of self-sterile species. The brush used to transfer pollenis inserted between siblings, handle down, in a pot. Inexpensive brusheswith plastic handles are preferable to those with wooden handles asmicroorganismal activity will cause the latter to rot. If pollen of adesired parent is unavailable from living plants, use pollen that wascollected at an earlier time and stored frozen in gelatin capsules.Gwynn has found that frozen pollen from N. tabacum may remain viable foras long as 7 years.

[0045] If a parental plant is suspected of harboring a mechanicallytransmitted virus, the instruments are sterilized in 70 percent ethanol.After a pollination has been made, a length of drinking straw, slightlylonger than the pistil, is placed over the stigma and crimped by foldingto prevent contamination by self- or foreign-pollen. Use of straws isprobably unnecessary if plants are well spaced on the greenhouse bench,mature blossoms are removed, and neither insects nor excessive airmovements present a problem. Furthermore, under most circumstances, astigma thoroughly covered with the intended pollen offers sufficientprotection against contamination. Usually, fertilization is affectedwithin 1 to 2 days. Mark individual crosses with a small, stringed keytag. Moisten the string before looping it around the pedicel otherwisethe flower may be severed when the string is tightened. Whenever anumber of identical crosses are planned, time can be saved by markingthe tags in advance of pollination.

[0046] Generally, seed of most Nicotiana spp. will mature about 28 daysafter pollination. Seedlings of N. tabacum may be obtained earlier byscraping the developing seeds from the carpels about 18 days afterpollination and treating them in 0.5 percent sodium hypochlorite (Cloroxand water, 1:9) for about 10 minutes. One or two capsules of N. tabacumwill contain enough seed to maintain a breeding line, but the seed yieldper capsule of other species may be considerably smaller.

[0047] Some Nicotiana spp. are essentially day neutral whereas othersrequire special temperature and day-length conditions to initiateflowering. Therefore, when planning specific hybridizations, considerthe day-length requirements of the species involved, unless you canfreeze pollen for use at a later time. Certain crosses may require amultitude of individual pollinations with the hope of obtaining a singleseed among many mature capsules, for example, the cross diploid N.rustica x diploid N. tabacum. Difficult hybridizations may yield apaucity of shrunken seed of a smaller than average size. Although mostseeds of this kind are often inviable, some of them may be induced togerminate if they are sown on soil in covered plastic or glasspreparation dishes. when seeds are limited in number, do not treat theseedlings with colchicine. Selecting the most vigorous plants is betterfor asexual propagation. Later, treat the growing points of the clonalmaterial with 0.5 percent aqueous colchicine three times a day for 3days to induce chromosome doubling.

[0048] Premature blossom drop may be avoided by the use of 0.5 percentindoleacetic acid in lanolin. Apply it by dipping a needle into thepreparation and scratching the pedicel at its point of attachment. Thistreatment will cause the blossom to remain on the plant regardless ofembryo development and, therefore, offers no guarantee of a successfulhybridization. On the other hand, syngamy serves to prevent abscission.

Example 2

[0049] Selection of Parental Lines for N. excelsior/N. benthamianaHybrids

[0050] Seven accessions of N. excelsior were collected. All of theaccessions had similar characteristics, but only TW46 and TW47 weredetermined to be good hosts for GFP Geneware™ viral vector. TW46 wasidentified as a poor host for certain insert genes in Geneware™ viralvector. Since systemic necrosis was observed in TW46, TW47 was selected.A group of over 100 N. excelsior TW47 were inoculated to give astatistically valid survey of the performance of GFP Geneware™ viralvector on N. excelsior. Almost all of the plants gave good-to-excellentsystemic GFP expression. A virus preparation from 200 grams of infectedwhole plants gave a final yield of 2 mg virion/g fresh weight.Unexpectedly this was 5- to 10-fold higher than the virion yieldtypically obtained from tobacco with CP-fusion constructs. The highervirion yield for N. excelsior when extended to the field, the yield peracre would be similar to tobacco, but the amount of tissue (waste) inthe bioprocessing stream is greatly reduced. Typical results for N.benthamiana are 0.9 mg virion/g fresh weight.

[0051] Seven accessions of N. benthamiana were screened for biomass andas a host for systemic expression from dual-subgenomic Geneware™ viralvector. One of the best performers was TW16.

Example 3

[0052] A primary interspecific hybrid between N. excelsior TW47 and N.benthamiana TW16 gave very good systemic GFP expression with GFPGeneware™ viral vector. Midvein regeneration was performed to stimulatechromosome-doubling and restoration of fertility (References: Campbellet al., Theor Appl Genet 87 (1994) 837-842; Kasperbauer and Collins,Crop Sci 12(1972)98-101). TW47 and TW16 Accessions were each obtainedfrom the USDA Tobacco Germplasm Collection in Oxford, N.C. Currentlythis collection is curated by Verne A. Sisson, Department of CropScience, North Carolina State University, Raleigh, N.C. 27695 (Phone:919/693/5151, extension 228).

[0053] Several regenerants set seed indicating chromosome-doubling andrestored fertility. The working name for the hybrid species is N.excelsiana. The seed was germinated, and the resulting plants wereevaluated for biomass and systemic Geneware™ viral vector expression.Systemic GFP expression was near the level of N. benthamiana. Biomassand growth habit were similar to N. excelsior.

[0054] Plants were grown in Kentucky for an agronomic test. The plantssurvived well in the field (better than the N. benthamiana) and showedsome bluemold resistance. Biomass was intermediate between the parentalvalues.

[0055] The primary chromosome-doubled hybrid between N. excelsior and N.benthamiana (N. excelsiana) appears to be a stable true-breeding line.It has good biomass and excellent Geneware™ viral vector properties, anddid well in a test in Kentucky.

[0056] In Table 1 listed below the phenotypic observations for severaltraits are shown for two lines each of the parents Nicotiana benthamianaTW16; Nicotiana excelsior TW47 and the hybrid Nicotiana excelsiana 7.b.TABLE 1 Phenotypic Observations Planted 3/10/99; Data Collected 4/22/99Nicotiana benthamiana Nicotiana excelsior Nicotiana excelsiana TraitsTW16 #1 TW16 #2 TW47 #1 TW47 #2 7.b #1 7.b #2 First flower open 4/23/994/22/99 Budding Budding Budding Budding Height (to top of stem) 20 cm  26 cm   27 cm 20 cm 34 cm 26 cm Largest leaf (Length) 18 cm 19.5 cm17-20 cm 19.5 cm   21 cm 19 cm Largest leaf (Width) 13 cm 13.5 cm 10-12cm 10 cm 14 cm 13 cm Leaf shape Round Round Oblong Oblong Oval Oval Leafangle 90° 90° 90° 90° 90° 90° Leaf color Med. green Med. green Darkgreen Dark green Med. green Med. green Trichomes Mod. #, small Mod. #,small Few, large Few, large Few, large Few, large Petiole leaf No NoYes, 70-80 mm Yes, 40 mm Yes, 20 mm Yes, 14 mm Petiole length  5 cm   6cm Continuous w/ Continuous w/  6 cm  5 cm leaf lamina leaf lamina Leaflamina 13 × 13 cm 13.5 × 13.5 20 × 12 cm 19.5 × 10 cm 15 × 14 cm 14 × 13cm cm Internode length 10 mm 15 mm 15 mm 15 mm 22 mm 21 mm Stalkdiameter  7 mm  7 mm  7 mm  8 mm  7 mm  7 mm Branching Yes @ every Yes @every Yes, barely Not yet Yes, 2 nodes Yes, 2 nodes leaf node leaf nodefrom 5 leaf starting to starting to nodes extend extend Flower form NbNb Flower color White White Flower head habit 1 flower/leaf 1flower/leaf node on main node on main stem stem

[0057] TABLE 2 Phenotypic Observations Planted 3/10/99; Data Collected4/26/99 Nicotiana benthamiana Nicotiana excelsior Nicotiana excelsianaTraits TW16 #1 TW16 #2 TW47 #1 TW47 #2 7.b #1 7.b #2 First flower openYes, secondary Yes, secondary 4/25/99 Buds on 4/25/99 Buds 4/25/99 BudsBudding; Estd meristems meristems topmost on topmost on topmost openingstarting bud starting bud branches branches branches 4/28/99 Height (totop of stem) 24 cm 31 cm 40 cm 31 cm 49 cm 40 cm Largest leaf (Length)21 cm 21 cm 21 cm 21 cm 23 cm 22 cm Largest leaf (Width) 15 cm 15 cm 14cm 11.5 cm   15.5 cm   13.7 cm   Leaf shape Round Round Oblong OblongOval Oval Leaf angle 90° 90° 90° <90° 90° 90° Leaf color Med. green Med.green Dark-med. green Med. green Med. green Med. green Trichomes Small,med. # Small, med. # Few, large Few, large Few, large Few, large Petioleleaf No No Yes, 9.5 cm Yes, 4 cm Yes, 20 mm Yes, 18 mm Petiole length 5cm 6 cm N/A N/A 7 cm 5.5 cm Leaf lamina 16 × 15 cm 15 × 15 cm 21 × 14 cm21 × 11.5 cm 16 × 15.5 cm 16.5 × 13.7 cm Internode length 1.6 cm (10 2cm (10 3.2 cm (5 2.9 cm (5 3 cm (8 2.4 cm (10 nodes) nodes) nodes)nodes) nodes) nodes) Stalk diameter 7 mm 8 mm 8 mm 7 mm 8 mm 8 mmBranching Yes, almost all Yes, almost all Yes, 5 are Yes, 5 are Yes, 9are Yes, 6 are leaf nodes leaf nodes extending extending extendingextending Flower form Nb Nb Large Large Medium N/A Flower color WhiteWhite White White White N/A Flower head habit 1/node from 1/node from1/node from 1/node from 1/node from N/A main stalk main stalk main stalkmain stalk main stalk

[0058] TABLE 3 Plant GFP-GJ GFP-pH5, Δ CP-GJ CP-pH5, Δ N. benthamiana0.5 0.6 1.1 1.6 lab strain mg/g FW mg/g FW mg/g FW mg/g FW N. tabacumMD609 ND ND ND ND BB/TT hybrid 0.2 0.1 0.2 0.1 N. benthamiana 1.0 0.81.4 1.2 TW16 N. excelsior TW47 1.2 0.8 2.6 2.4 N. excelsiana 0.8 0.6 1.92.2 N. occidentalis 0.4 0.6 1.5 1.7 TW91 N. umbratica 0.9 0.9 1.0 1.0TW144 TW91 × TW144 0.5 0.5 0.8 0.9 TW144 × TW91 0.7 0.6 1.3 1.3

Example 4

[0059] Backcross Progeny from Interspecific Hybrid TW47 x TW16

[0060] Backcrosses to each parental type were performed in order toevaluate the characteristics of the sesquidiploids. The backcross to N.excelsior was the only one to germinate. They looked much like N.excelsior and gave a similar GFP expression pattern to N. excelsior. Anadditional backcross was performed to generate the first breakdowngeneration, and the resulting plants are currently being evaluated.

Example 5

[0061] In-field screening of various Nicotiana species was continued in1999. The purpose of this study was to evaluate biomass differencesbetween species, to evaluate growth characteristics differences betweenspecies, and to evaluate insect and disease pressure differences betweenspecies.

[0062] Nine different Nicotiana species were seeded in the greenhouse onApril 14. Each species was seeded into three 72-cell trays and placed ina specially constructed float bed. Burley Gold growth media was used forthe transplants. Trays were filled manually and dibbled using the GolfBall Dibbler. Raw seed was used for eight of the species and pelletedBenthamiana seed was used for the lab strain Benthamiana transplants.The GROmore hand seeder was used to sow the raw seed and the pelletedseed was sown by hand. Each tray was labeled with a tray marker toidentify each species. The pelleted lab strain Benthamiana trays werewatered overhead for three days post-seedling to dissolve the claypellet coating and facilitate cotyledon emergence. Table 4 outlines thespecies transplanted in this study. TABLE 4 Nicotiana SpeciesTransplanted Nicotiana Species Species Identifier TW 17-NicotianaBenthamiana Harvested 12/8/97 TW 16-Nicotiana Benthamiana Harvested11/26/97 SCR1-Nicotiana Benthamiana Harvested 11/6/98 L3663-NicotianaBenthamiana Harvested 4/4/98 Nicotiana Excelsiana Lot #7.b, Harvested5/22/98 Nicotiana Occidentalis Obliqua Unknown SCRI Stock TW47-Nicotiana Excelsior Harvested 3/31/98 TW 91-Nicotiana OccidentalisHarvested 12/16/97 Lab Strain-Nicotiana Benthamiana Lot #28b 29b RC

[0063] Transplants were treated, through the float water, with thefungicide Ridomil. An insecticide, Orthene, and a foliar appliedfungicide, Dithane were also applied to the transplants. At 21 DPT,fertilizer was applied to the float water. A fertility level of 7 on theDiSST-4 meter was maintained throughout transplant production.

[0064] Transplants were transported to the ARP for transplanting on May20, 37 DPS. Plants were transplanted on raised, black plastic coveredbeds, using a mechanical transplanter at a density of four plants perlinear foot of bed. The plants were transplanted in two rows on the bedwith an offset plant configuration. The transplant water contained ½gallon of 7-14-7 Starter Fertilizer and ½ pound of Orthene per 150gallons of water. Each species was replicated three times with eachreplication consisting of ten feet of bed.

[0065] At transplanting, several differences were apparent in transplantquality. TW 16 produced a slightly leggy transplant that wilted soonafter transplanting. TW 17 transplants were smaller than the otherBenthamiana transplants. However, TW 17 transplants had a thicker stemand appeared much hardier than the other Benthamiana transplants. TheLab Strain Benthamiana plants were much smaller and less consistent inplant size due to problems with germination during the transplantproduction phase. These problems were probably due to the fact thatthese seeds were pelleted. Some of the Obliqua transplants were alreadyelongated at the time of transplanting. The highest quality transplantswere the Excelsiana seedlings. These seedlings were very dark green incolor, held a thick stem, and appeared to handle transplant stress muchbetter than the other seedlings in this plot.

[0066] All plot replications were treated every 7-10 days with Ortheneand Dithane at a rate of 1 teaspoon/gallon. Soil moisture was monitoredusing gypsum blocks that were buried about 4-5 inches in the beds. Noirrigation was necessary for this plot due to the timely rainfall.

[0067] On June 29, 43 DPT, four plants from each replication weresampled for fresh weight determination. Table 5 illustrates the resultsfor each replication. TABLE 5 Fresh Weights for each Replication SpeciesReplication No. Weight/Plant (g/plant) TW 16 Benthamiana 1 411.6 TW 16Benthamiana 2 235.7 TW 16 Benthamiana 3 280.1 TW 17 Benthamiana 1 426.8TW 17 Benthamiana 2 352.6 TW 17 Benthamiana 3 Dead SCRI Benthamiana 1326.3 SCRI Benthamiana 2 236.6 SCRI Benthamiana 3 Dead L3663 Benthamiana1 335.6 L3663 Benthamiana 2 292.1 L3663 Benthamiana 3 Dead TW 47Excelsior 1 616.5 TW 47 Excelsior 2 450.6 TW 47 Excelsior 3 517.8 TW 91Occidentalis 1 528.8 TW 91 Occidentalis 2 399.8 TW 91 Occidentalis 3449.2 Occidentalis Obliqua 1 141.9 Occidentalis Obliqua 2 620.6Occidentalis Obliqua 3 809.7 Excelsiana 1 333.0 Excelsiana 2 512.4Excelsiana 3 580.8 Lab Strain Benthamiana 1 90.5 Lab Strain Benthamiana2 200.5 Lab Strain Benthamiana 3 N/A

[0068] Several replications died due to root rot induced by excess watersurrounding the raised bed. This excess water came about due toexcessive rainfall and poor plot drainage.

[0069] Table 6 summarizes the replication plant weights. The followingTable 6 is the average of all replications for each species. TABLE 6Replication Averages by Species Species Average Plant Weight (g/plant)TW 16 Benthamiana 305.8 TW 17 Benthamiana 389.7 SCRI Benthamiana 281.5L3663 Benthamiana 313.9 TW 47 Excelsior 528.3 TW 91 Occidentalis 459.3Occidentalis Obliqua 524.1 Excelsiana 475.4 Lab Strain Benthamiana 145.5

[0070] TW 17 performed well in comparison to the other Benthamiana inthe plot. TW 17 was a very vigorous plant with excellent hardiness andhigh biomass levels. More work should be done to determine thefeasibility of this plant as a viral vector host. Also, Excelsiana stoodout as a superior plant in this plot. This plant offered high biomasslevels with significant leaf size and weight.

[0071] This invention is also directed to methods for producing aNicotiana plant by crossing a first parent Nicotiana plant with a secondparent Nicotiana plant, wherein the first or second Nicotiana plant isthe Nicotiana plant from the line Nicotiana Interspecific Hybrid.Further, both first and second parent Nicotiana plants may be from thecultivar TW47 x TW16. Therefore, any methods using this cultivar arepart of this invention: selfing, backcrosses, hybrid breeding, andcrosses to populations. Any plants produced using cultivar as a parentare within the scope of this invention.

[0072] As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which Nicotiana plantscan be regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants, such as pollen, flowers, embryos,ovules, seeds, pods, leaves, stems, roots, anthers and the like. Thus,another aspect of this invention is to provide for cells which upongrowth and differentiation produce a cultivar having essentially all ofthe physiological and morphological characteristics of NicotianaInterspecific Hybrid.

[0073] Culture for expressing desired structural genes and culturedcells are known in the art. Also as known in the art, Nicotiana aretransformable and regenerable such that whole plants containing andexpressing desired genes under regulatory control may be obtained.General descriptions of plant expression vectors and reporter genes andtransformation protocols can be found in Gruber, et al., “Vectors forPlant Transformation, in Methods in Plant Molecular Biology &Biotechnology” in Glich, et al., (Eds. pp. 89-119, CRC Press, 1993).Moreover GUS expression vectors and GUS gene cassettes are availablefrom Clone Tech Laboratories, Inc., Palo Alto, Calif. while luciferaseexpression vectors and luciferase gene cassettes are available from ProMega Corp. (Madison, Wis.). General methods of culturing plant tissuesare provided for example by Maki, et al., “Procedures for IntroducingForeign DNA into Plants” in Methods in Plant Molecular Biology &Biotechnology, Glich, et al., (Eds. pp. 67-88 CRC Press, 1993); and byPhillips, et al., “Cell-Tissue Culture and In-Vitro Manipulation” inCorn & Corn Improvement, 3rd Edition; Sprague, et al., (Eds. pp.345-387) American Society of Agronomy Inc., 1988. Methods of introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant cells with Agrobacterium tumefaciens, Horsch etal., Science, 227:1229 (1985). Descriptions of Agrobacterium vectorssystems and methods for Agrobacterium-mediated gene transfer provided byGruber, et al., supra.

[0074] Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using the microprojectile media delivery with the biolisticdevice Agrobacterium-medicated transformation. Transformant plantsobtained with the protoplasm of the invention are intended to be withinthe scope of this invention.

[0075] The present invention contemplates a Nicotiana plant regeneratedfrom a tissue culture of a variety or hybrid plant of the presentinvention. As is well known in the art, tissue culture of Nicotiana canbe used for the in vitro regeneration of a Nicotiana plant. Tissueculture of various tissues of Nicotiana and regeneration of plantstherefrom is well known and widely published. For example, reference maybe had to Weissbach and Weissbach (eds.), Meth. Enzymol. 118 (1986);Zaitlin et al. (eds), Biotechnology in Plant Science (1985).

[0076] When the term Nicotiana plant is used in the context of thepresent invention, this also includes any single gene conversions ofthat variety. The term single gene converted plant as used herein refersto those Nicotiana plants which are developed by a plant breedingtechnique called backcrossing wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single gene transferred into the varietyvia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into thevariety. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent. The parentalNicotiana plant which contributes the gene for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental Nicotiana plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehlman &Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the originalvariety of interest (recurrent parent) is crossed to a second variety(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until aNicotiana plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

[0077] The selection of a suitable recurrent parent is an important stepfor a successful backcrossing procedure. The goal of a backcrossprotocol is to alter or substitute a single trait or characteristic inthe original variety. To accomplish this, a single gene of the recurrentvariety is modified or substituted with the desired gene from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological, constitution of the original variety. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross, one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

[0078] Many single gene traits have been identified that are notregularly selected for in the development of a new variety but that canbe improved by backcrossing techniques. Single gene traits may or maynot be transgenic, examples of these traits include but are not limitedto, male sterility, herbicide resistance, resistance for bacterial,fungal, or viral disease, insect resistance, male fertility, enhancednutritional quality, industrial usage, yield stability and yieldenhancement. These genes are generally inherited through the nucleus.

DEPOSIT INFORMATION

[0079] A deposit of the Nicotiana seed of this invention has been madewith the American Type Culture Collection, Manassas, Va. The deposit wasmade on Jul. 12, 1999 and has ATCC Accession No. PTA-323.

[0080] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

1. An interspecific Nicotiana hybrid of Nicotiana Excelsior x N.Benthamiana, wherein a sample of said seed has been deposited under ATCCAccession No. PTA-323.
 2. A plant, or its parts, produced by growing theseed of claim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule of theplant of claim
 2. 5. A Nicotiana plant having essentially all of thephysiological and morphological characteristics of the Nicotiana plantof claim 2, or its parts.
 6. Tissue culture of the seed of claim
 1. 7. ANicotiana plant regenerated from the tissue culture of claim
 6. 8.Tissue culture of regenerable cells of the plant, or its parts, of claim2.
 9. The tissue culture of claim 8 wherein the regenerable cells areembryos, meristematic cells, pollen, leaves, anthers, roots, root tips,flowers, seeds, stems, pods, or protoplasts or calli derived therefrom.10. A Nicotiana plant regenerated from the tissue culture of claim 9.11. A method of producing backcross progeny from interspecific Nicotianahybrids comprising: a. selecting one parent of an interspecific hybridas a recurrent parent based on biomass greater than the primaryinterspecific hybrid; b. backcrossing said interspecific hybrid to therecurrent parent and collecting the resulting seed.
 12. The seedproduced from the method of claim
 11. 13. A Nicotiana plant, or itsparts, produced by growing the seed of claim
 12. 14. Tissue culture ofregenerable cells of the plant, or its parts, of claim 13, wherein theregenerable cells are selected from the group consisting of: embryos,meristematic cells, pollen, leaves, anthers, roots, root tips, flower,seeds, stems, protoplasts and calli derived therefrom.
 15. A method ofproducing backcross progeny from interspecific Nicotiana hybridscomprising: a. selecting one parent of an interspecific hybrid as arecurrent parent based on capacity for high level expression of systemicprotein from a viral vector; b. backcrossing said interspecific hybridto the recurrent parent and collecting the resulting seed.
 16. The seedproduced from the method of claim
 15. 17. A Nicotiana plant, or itsparts, produced by growing the seed of claim
 16. 18. Tissue culture ofregenerable cells of the plant, or its parts, of claim 17, wherein theregenerable cells are selected from the group consisting of: embryos,meristematic cells, pollen, leaves, anthers, roots, root tips, flower,seeds, stems, protoplasts and calli derived therefrom.